Immunoassay is a class of scientific techniques used to detect small amounts of biomolecules in biological samples. These techniques are widely used in medical diagnostic procedures, such as blood tests and the like, to detect specific proteins ("antigens"). Conventional methods of immunoassay rely on the selective reaction of a particular protein with an antigen. For example, it is well known that animals develop a specific antibody to combat a particular foreign antigen. Because the antibody has a highly specific chemical configuration, it reacts selectively with the antigen and not with other proteins. The reaction of the antibody with the antigen ("precipitation") binds the antigen so that it can be further processed and, ultimately, destroyed or removed from the system. Such antibody-antigen reactions give rise to the well-known immune response in animals, by which animals are able to resist infections to which they have been previously exposed.
This ability of antibodies to selectively bind to antigens can also be used to detect antigens present in a biological sample. For example, when a patient has a particular illness, one or more characteristic antigens will be present in the blood. An antibody to that antigen will selectively bind to the antigen. A common type of immunoassay which relies on this effect is the "antibody capture assay." To detect an antigen in a particular sample, the antigen is first immobilized by attaching it to a solid support. A support often used for this purpose is a "microtiter plate," which is a plastic plate made of clear polystyrene or polyvinylchloride. The plate is molded with 96 depressions or wells in which biological samples are held for testing. When a sample is added to a well in the plate, antigens in the sample are bound or attached to the support, and are thereby immobilized. This binding is not a chemical reaction but, rather, is believed to result from a physical or noncovalent interaction between the polystyrene matrix and the antigen.
Once the antigen is bound to the support, an antibody known to be specific to that antigen is allowed to react with, and bind to, the immobilized antigen. It is then possible to detect the presence of the antigen indirectly by detecting the antibody bound to the antigen. This is accomplished by "labelling" the antibody. One way antibodies are labelled is with a radioactive isotope, which is incorporated into the molecular structure of the antibody, and can be detected using known detection systems.
Another commonly used labelling method is to bind a second antibody to the first antibody. This second antibody is linked with a "marker" enzyme, such as horseradish peroxidase. A dye which is substantially water-insoluble and changes color in the presence of the marker enzyme is added to the solution. Thus, if sufficient antigen is bound to the walls of the microtiter plate, it is possible to detect the antigen by the color change which occurs when the dye reacts with the marker enzyme.
Within limits, it is also possible to determine how much antigen is present by measuring the intensity of this color change. One well known technique which relies on this effect is ELISA (Enzyme Linked Immunosorption Assay). With ELISA, the amount of antigen present is determined by measuring the amount of color change in the wells of the microtiter plate using a photometer known as an "ELISA reader." A detailed description of ELISA techniques can be found in E. Macy, et al., "Enhanced ELISA: How to measure less than 10 picograms of a specific protein in less than 8 hours, . . . "F.A.S.E.B. Journal, Vol 2, pp. 3003-009 (1988), and S. F. De St. Groth, "The evaluation of limiting dilution assays," J. Immunol. Meth., Vol. 49, pp. R11-R23 (1982), both of which are incorporated herein by reference and made a part hereof.
Because ELISA is based on measuring the color change caused by the reaction of a dye with the marker enzyme in a solution, it has limited sensitivity. When small amounts of antigen are present, the color change is so slight that it can be lost in the background "noise" of the measurement system. Noise is created when excess antibodies or marker enzymes bind directly to the walls of the support. When the dye is added, these materials cause a color change which is not related to the amount of antigen present. In addition, once a certain amount of color change occurs, further color change is difficult to detect, making the assay less than ideal for quantifying larger amounts of antigen.
Most significantly, because ELISA relies on the detection of light which passes through the stained solution, only clear supports--which do not interfere with the passage of light--can be used to bind the antigen. The need for a transparent support places limitations on the surface characteristics of the support materials which may be used for ELISA and similar assays. Thus, if a particular support material does not have the desired optical transparency, it cannot be used, even if it has better antigen binding properties.
To date, attempts to increase the sensitivity of ELISA have concentrated on increasing the binding capacity of the support without diminishing its optical transparency, so that the sample can be analyzed photometrically. Several such attempts have been directed to changing the chemical configuration of the surface so that it will form a chemical bond with the antigen. For example, U.S. Pat. No. 4,933,410 to Okrongly discloses activating polystyrene supports by reacting the surface of the support with hydroxymethylamides in a polystyrene insoluble solvent. It is recommended that this activation take place under mild conditions in a solvent which neither dissolves nor swells the polystyrene to maintain the optical clarity of the support. Similarly, U.S. Pat. No. 4,119,589 discloses activating a compound having at least two secondary amine groups by converting the secondary amine groups into imino-chloride groups.
It has also been proposed to add a coating to the surface of the support to bind the protein. For example, U.S. Pat. No. 4,210,418 to Brown, et al. discloses coating the support with an inert protein which binds the antigen by adsorption, ionic binding, entrapment or, more preferably, covalent binding. To facilitate attachment of the inert protein, the surface of the support may be treated with materials, including solvents, surfactants, acids, or bases, to enhance adsorption. (See U.S. Pat. No. 4,210,418, col. 5, Ins. 12-16.)
The binding of biomolecules such as antigens to solid supports has three basic components: capacity, affinity, and stability. Capacity is the maximum amount of material that can be bound per surface area of support. Affinity is the degree of attraction between the antigen and the support. Stability is the level of permanence of the bond between the biomolecule and the support. While the affinity and stability of binding is thought to be an inherent feature of the particular polymeric material being used, capacity can be enhanced by activation processes.
The methods of activating supports described above have focused on changing the surface properties of the support in an effort to increase binding capacity. However, merely increasing binding capacity does not ensure that the support will be better for immunoassay. For example, if the stability of binding is low, the bound biomolecules will tend to bleed off during biochemical analysis, decreasing the sensitivity of the analysis. Likewise, if the affinity of the support is low, binding will be too slow--a disadvantage for analytical procedures.
Other supports currently used for immunoassay include porous membranes made of nitrocellulose, nylon, and similar materials. These materials are porous and hydrophilic, that is, they tend to bind or absorb water. The use of such hydrophilic materials is thought to increase binding capacity by drawing water (in which the biomolecules are carried) into the support. Although such supports have improved binding characteristics, they suffer from several drawbacks. First, because these materials are hydrophilic, samples blotted onto such supports spread and do not remain on the surface of the support. Therefore, it is difficult to saturate free binding sites on the support. Antibodies, dyes, and other materials used for detection can bind to these available sites, resulting in false readings or background staining. Also, because these supports are hydrophilic, washing does not remove these materials as completely or efficiently; as a result, it is more difficult to accurately quantify the amount of a particular biomolecule that is present.
Accordingly, it is an object of the present invention to provide a method of performing an assay for biomolecules which is more accurate than conventional methods.
It is a further object of the invention to provide an assay method which can be performed using readily available materials and comparatively inexpensive detection equipment.
It is a further object of the invention to provide an assay method in which biomolecules can be detected using water-insoluble dyes.
It is a further object of the invention to provide an assay method in which background staining and resultant noise can be significantly reduced, thereby increasing the accuracy and sensitivity of the assay.
It is a further object of the invention to provide an assay method which can be relatively easily performed with a minimum of labor-intensive steps.
It is a further object of the invention to provide an assay method using solid supports which do not have to be optically transparent.
It is a further object of the invention to provide a solid support for use in detecting the presence of biomolecules which has improved binding capacity and affinity.
It is a further object of the present invention to provide a solid support which is not hydrophilic so that the binding of biomolecules to the support may be carefully controlled.
It is a further object of the invention to provide a solid support which is made using readily available, inexpensive materials and relatively simple production processes.